Characterization of a cathepsin L-like enzyme secreted from human pancreatic cancer cell line HPC-YP.
Spent culture medium from the human pancreatic carcinoma cell line HPC-YP, which can propagate in a protein-free, chemically defined medium without any other supplements, was analyzed for the presence of the cysteine protease, cathepsin L. The secreted form of cathepsin L was distinguished from the lysosomal form by its increased stability at alkaline pH, by its strong thermostability, and by its larger molecular size. HPC-YP cathepsin L was still stable at pH 7.4 and at 56 degrees C after 60-min preincubation. The molecular weight of this enzyme was estimated to be 68,000, compared with a molecular weight of 29,000 for normal liver cathepsin L. By Western blot analysis, HPC-YP enzyme was found to be composed of two components, one with a molecular weight of 37,000 and the other of 31,000. This result suggests that HPC-YP enzyme in the spent medium may be a complex of the proenzyme (in the case of liver proenzyme; Mr 39,000) and the mature enzyme (in the case of liver mature enzyme; Mr 29,000). Interestingly, an intrinsic inhibitor was also separated from the spent medium by gel filtration. The molecular weight of this inhibitor was estimated to be approximately 13,000. The cathepsin L of HPC-YP proved more resistant toward leupeptin than did liver cathepsin L. On the other hand, the former was more sensitive than the latter toward the diazomethane inhibitors, Z-Phe-Phe-CHN2 and Z-Phe-Ala-CHN2. These results indicate that cathepsin L secreted from cancer cell lines may play a role in the destruction of basal lamina, invasion of tissue, and formation of metastasis.
Yamaguchi
N
N
Department of Microbiology, Kyoto Prefectural University of Medicine, Japan.
Chung
S M
SM
Shiroeda
O
O
Koyama
K
K
Imanishi
J
J
D015153:Blotting, Western; D002277:Carcinoma / Q000201:enzymology*; D056668:Cathepsin L; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D003546:Cysteine Endopeptidases; D010450:Endopeptidases; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D010190:Pancreatic Neoplasms / Q000201:enzymology*; D011480:Protease Inhibitors / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D011480:Protease Inhibitors; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; C534281:CTSL protein, human; D056668:Cathepsin L